University of Huddersfield Repository Shokry, Dina Predicting Human Intestinal Absorption Using Chromatography and Spectroscopy Original Citation Shokry, Dina (2017) Predicting Human Intestinal Absorption Using Chromatography and Spectroscopy. Doctoral thesis, University of Huddersfield. This version is available at http://eprints.hud.ac.uk/id/eprint/34142/ The University Repository is a digital collection of the research output of the University, available on Open Access. Copyright and Moral Rights for the items on this site are retained by the individual author and/or other copyright owners. 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For more information, including our policy and submission procedure, please contact the Repository Team at: [email protected]. http://eprints.hud.ac.uk/ PREDICTING HUMAN INTESTINAL ABSORPTION USING CHROMATOGRAPHY AND SPECTROSCOPY Dina Shokry A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Under supervision of Dr Laura J Waters Dr Gareth M Parkes School of Applied Sciences The University of Huddersfield 2017 Dedication This thesis is dedicated to my dear loving parents, you were, still are and always will be my backbone in life. Without your love, support and belief in me none of this would have been possible. ii Acknowledgement I would like to praise God for his countless blessings and giving me the strength and power to complete my work and achieve my goals. Also I would like to show my deep and sincere appreciation to my main supervisor Dr Laura Waters for her endless support, understanding and concern on both the professional and personal level. Without her scientific and technical guidance, her patience and continuous encouragement, none of this would have been possible. I would also like to express my profound gratitude to my second supervisor Dr Gareth Parkes for his help, valuable advice, guidance and support. I would also like to thank Professor John C. Mitchell for his valuable guidance throughout the publishing of one of the thesis papers. Also I would like to thank the University of Huddersfield Graduate Centre for providing me with the needed conference presentation funds during the time of my PhD. I must also extend my thanks to the technical staff of Huddersfield University, School of applied sciences for their great help throughout the duration of my PhD and to my friends and colleagues who made my PhD time an enjoyable time. I would like to thank my parents for their unconditional love and genuine concern and always believing in me and making me feel I am not alone. Thank you for always pushing me forward, supporting and helping me, no words can thank you enough, I am really blessed to have you as my parents. Finally, I would like to thank my sisters who have always been there for me whenever I needed their support, encouragement and wise advice. Also I thank my nephew and niece, the two little angels, who always put a smile on my face. iii Abstract New drug entities (NDE) are constantly being developed with most of them intended for oral administration. For this reason, there is a need to estimate their absorption in order to save time and money that would be lost if the drug enters the clinical stage and is then found to exhibit poor absorption. For many years, the use of animals was the most abundant method for studying pharmacokinetics to predict parameters such as intestinal absorption. However, these methods are time consuming, and expensive as well as being ethically unfavourable. As a result, developing other methods to evaluate a drug’s pharmacokinetics is crucial. The aim of this work was to develop in vitro methods for estimation of human intestinal absorption (%HIA) to replace the use of the aforementioned, less favourable methods involving the use of animals. Among the developed methods in this thesis is a unique type of chromatography known as micellar liquid chromatography (MLC) using biosurfactants such as bile salts as a mobile phase. Furthermore, studies investigated the effect of a change in the stationary phase in addition to investigating the effect of the change in temperature on 2 the elution of the analysed compounds. It was found that R PRED for the developed MLC methods was in the range of 43.3 % - 91.12 %. Another developed method was a spectrophotometric method based on the use of the solubilising effects of bile salts, as well as their binding to compounds. Therefore, two spectrophotometric methods were developed, a solubilisation method and a double reciprocal method, and used in the prediction of %HIA. It was found that the solubilisation method had a better 2 predictability for %HIA than that of the double reciprocal method where R PRED was found to be 82.32 % and 61.90 % respectively. Finally, a permeation method was developed using the ability of NaDC to form a hydrogel under specific conditions and applying the investigated drugs in an infinite dose to the prepared hydrogels. This facilitated the determination of permeability coefficients (Kp) that were then used in the prediction of %HIA using the obtained model. The two developed permeation methods 2 2 were found to have close values of R PRED for % HIA where R PRED of the permeation method using flow through cells was found to be 79.8 % while that of the permeation method using Franz cells was found to be 79.67 %. In summary, this work reports several unique models for the in vitro prediction of human intestinal absorption, potentially removing the need for animal testing to predict %HIA. iv List of Abbreviations Abbreviation Full term IAMs Immobilised Artificial Membranes. ILC Immobilised Liposome Chromatography. CMC Critical Micellar Concentration. HPLC High performance liquid chromatography TLC Thin layer chromatography GC Gas chromatography MLC Micellar Liquid Chromatography IP Ion Pairing. BMC Biopartitioning Micellar Chromatography QSAR Quantity structure activity relationship NaDC Sodium deoxycholate NaTDC Sodium taurodeoxycholate NaC Sodium cholate NaTC Sodium taurocholate SIFsp Simulated Intestinal Fluid sine pancreatine CM Micellar concentration %HIA % Human Intestinal Absorption CE Capillary electrophoresis MEKC Micellar electrokinetic chromatography MEEKC Microemulsion electrokinetic chromatography CCC Counter current chromatography PSA Polar Surface Area nHA Number of hydrogen bond acceptors nHD Number of hydrogen bond Donors FRB Free Rotatable bonds MLR Multiple linear regression MLP Molecular lipophilicity potentials Ko/w octanol-water partition coefficient SEM Scanning Electron Microscopy FT-IR Fourier Transform Infrared v List of Contents Chapter 1: Introduction and Literature Review .................................................................... 1 1. Introduction .................................................................................................................... 1 1.1. Drug development and Intestinal permeability: ........................................................... 1 1.2. Mechanisms of permeation of compounds across intestinal membrane:..................... 1 1.2.1. Passive transcellular diffusion: ............................................................................. 2 1.2.2. Paracellular passive transport: ............................................................................. 3 1.2.3. Carrier-mediated transport: .............................................................................. 3 1.2.3. a. Active and facilitated transport: .............................................................................. 3 1.2.3. b. Receptor-mediated transcytosis: ............................................................................ 3 1.2.3. c. Efflux mechanism: ..................................................................................................... 3 1.3. Methods for determination of intestinal permeability: .................................................. 4 1.3.1. Cell culture based models: e.g. (Caco-2 cells) ................................................. 4 1.3.2. Membrane based models:................................................................................ 6 1.3.3. Ex Vivo models: ............................................................................................... 7 1.3.4. In Situ intestinal perfusion models .................................................................... 8 1.3.5. Everted intestinal ring/sac ................................................................................ 9 1.3.6. In silico models for prediction of intestinal permeability through in vitro-in vivo correlation ...................................................................................................................... 9 1.4. Importance of lipophilicity in medicinal chemistry and drug discovery: ...................... 10 1.5. Methods for determination of a partition coefficient: .................................................. 13 1.5.1. Direct methods: ..................................................................................................... 13 1.5.1. a. Shake Flask Method: .....................................................................................